The present invention concerns compounds that have high affinity for the imidazoline receptor, particularly the imidazoline I2 receptor, methods of use thereof, and combinatorial libraries thereof.
In the early 1980""s while studying the different hypotensive effects of imidazoline compounds vs. catecholamines in the nucleus reticularis lateralis, Bousquet et al. proposed the existence of a class of imidazoline (I) binding sites distinct from adrenergic receptors (Bousquet, P. et al., The Journal of Pharmacology and Experimental Therapeutics 230:232-236 (1984)).
Many subsequent studies have supported the presence of these binding sites in a variety of tissues (Coupry, I. et al., Biochemical and Biophysical Research Communications 147:1055-1060 (1987); Ernsberger, P. R. et al, European Journal of Pharmacology 134:1-13 (1987); Meeley, M. P. et al., Life Sciences, 38:1119-1126 (1986); Parini, A. et al. Journal of Biological Chemistry 264(20): 1874-8 (1989)), including the existence of the putative endogenous ligand (Atlas, D., and Y. Burstein, European Journal of Biochemistry 144:287-293 (1984); Li, G. et al., Science 263:966-969 (1994)) and purification of a mitochondrial imidazoline binding site (Limon, I. et al., Journal of Biological Chemistry 267(30):21645-9 (1992)).
The binding sites are classified into two groups, I1 and I2, according to their affinity for clonidine and idazoxan, respectively, and are located on the plasma and outer mitochondrial membranes. I2 binding sites are associated with the monoamine oxidase protein in certain tissues and are further sub-classified into I2A and I2B depending on their sensitivity to the compound amiloride (Regunathan, S., and D. J. Reis, Annual Review of Pharmacology and Toxicology 36:511-44 (1996)). A third subgroup classified as Iatypical have been identified in the pancreatic xcex2-cell (Morgan, N. G. et al., Annals of the New York Academy of Sciences 763:361-73 (1995); Brown, C. A. et al., British Journal of Pharmacology 108:312-317 (1993); Chan, S. L., Clinical Science 85(6):671-7 (1993)).
The I1 binding sites are localized on the plasma membrane and have been found in the brainstem, kidneys, adrenal chromaffin cells, rat PC12 cells and platelets. Imidazoline I1 receptor agonists which stimulate I1 receptors in the medulla offer a new therapeutic approach to the treatment of hypertension and associated metabolic syndrome comprising of hyperglycemia, hyperinsulinemia, hypertriglyceridemia and low concentrations of high-density lipoprotein cholesterol (Krentz, A. J., and A. J. Evans, The Lancet 351:152-153 (1998)). Selectivity for this receptor reduces the adverse effects attributed to stimulation of xcex12-adrenoceptors such as sedation and dry mouth. Platelet I1 receptors are upregulated in patients suffering with depression and can be downregulated with antidepressant therapy.
I2 binding sites are more widely distributed than I1 sites. They have been found in the nervous system (cerebral cortex, astrocytes, carotid bodies), specific organs (kidneys, liver, pancreas, colon, urethra, placenta), glands (adrenal medulla, prostate) and certain cell types (platelets, adipocytes, vascular cells). Expression of I2 binding sites is upregulated in human brains during aging, and in patients with Alzheimer""s disease. Expression is modified in the brains of depressed suicide victims (Regunathan, S., and D. J. Reis, Annual Review of Pharmacology and Toxicology 36:511-44 (1996)).
Efforts have been made to identify compounds with imidazoline receptor binding affinity:
U.S. Pat. No. 5,726,197 to Clark et al. describe isoindolinyl derivatives that have imidazoline receptor binding affinity.
U.S. Pat. No. 5,686,477 to Jarry et al. describes various 5-(aryloxymethyl)oxazolines having imidazoline binding activity.
U.S. Pat. No. 5,354,769 to Garcia Sevilla et al. describes benzofuranyl imidazole derivatives having imidazoline receptor binding activity, and various uses thereof.
U.S. Pat. No. 5,732,717 to Watanabe et al. describes the use of 4-chloro-5-(imidazoline-2-ylamino)-6-methoxy-2-methylpyrimidine for treating substance abuse withdrawal.
U.S. Pat. No. 5,574,059 to Regunathan et al. describes the treatment of vascular smooth muscle cell proliferation with certain imidazoline receptor agonists.
Nevertheless, there are relatively few compounds with imidazoline receptor binding affinity available, and there is continued interest in identifying additional imidazoline receptor binding compounds.
A first aspect of the present invention is a method of binding the imidazoline receptor, comprising contacting a bis-benzene to said imidazoline receptor in an amount effective to bind to said receptor, wherein said bis-benzene contains at least one amidine group. The amidine group is covalently bound to one of the benzene rings, or an amidine group is covalently bound to both of the benzene rings (such compounds and their pharmaceutically acceptable salts are referred to as xe2x80x9cactive agentsxe2x80x9d herein). The contacting step may be carried out in vitro (e.g., with cells that express the imidazoline receptor, or with a cell-free preparation comprising the imidazoline receptor) or in vivo (e.g., by administering said compound to a subject afflicted with a disease state which is alleviable by treatment with a compound having high selectivity and affinity for the imidazoline receptor site).
A second aspect of the present invention is a method of identifying imidazoline receptor binding agents, comprising the steps of providing a library of bis-benzene compounds, said bis-benzene compound containing at least one amidine group as described above; and screening said library for compounds that bind to said imidazoline receptor. The library may be a combinatorial library, and the bis-benzene compounds may be immobilized on a solid support in accordance with known techniques (e.g., where only one of the benzene groups has an amidine group covalently bound thereto, the other benzene group may be affixed, e.g., by covalent bond, to the solid support).